The proposed project is a unique synergy between basic and clinical research teams and contains the requisite sophistication to evaluate the novel concept of intestinal metabolic reprogramming as one of the key mechanisms of action of Roux-en-Y Gastric Bypass Surgery (RYGBS). Over the last decade, research has shown that RYGBS is the best treatment option for obesity-related type 2 diabetes (T2DM). The most exciting advance would be to reverse engineer RYGBS; that is to unravel the molecular mechanisms by which RYGBS exerts its metabolic effects and then to produce those effects without surgery. Understanding why a number of patients with T2DM who underwent RYGBS do not go into remission would ultimately improve patient management. Experiments in animal models and small pilot human studies have emphasized the role of the transposed intestine (Roux limb) as the key anatomic substrate of the mechanisms of metabolic improvement after RYGBS. In both rodents and human patients, the Roux limb increases its fuel utilization in an attempt to accommodate its increased bioenergetic requirements. Morphologically, this adaptive response appears as increased cellular proliferation and cytoskeletal/brush border remodeling. Metabolically, it manifests as increased sequestration and utilization of glucose, cholesterol and amino acids to support growth and tissue maintenance. This project will tackle several challenges and limitations currently hindering progress. It will enhance our understanding about the nature and the timing of intestinal adaptive changes, which are currently largely unknown. The serial assessment of intestinal metabolism using intestinal biopsies derived from subjects at the time of and 1, 6 and 12 months after RYGBS, will substantially facilitate the study of intestinal biology after RYGBS. This is currently hindered by limited availability of post-RYGBS intestinal samples. A further benefit of these studies is the premier establishment of a system that will allow the comprehensive examination of the effects of RYGBS on intestinal crypts and intestinal stem cells, using human intestinal organoids known as mini-guts. Another obstacle of progress in this field is the lack of in vivo studies focusing on intestinal glucose metabolism. Studies under the proposed project will quantify intestinal glucose utilization, with PET-MRI scanning with [18F]FDG, before and after RYGBS, and will compare elaborate quantitative and simplified semi-quantitative algorithms of intestinal glucose uptake. Finally, a targeted metabolite profiling of serum samples of subjects with T2DM, who participated in the Longitudinal Assessment of Bariatric Surgery (LABS) study, will allow us to examine whether markers of intestinal adaptation could serve as predictors of remission of T2DM after RYGBS. Overall, the proposed project will facilitate our understanding of the mechanisms of RYGBS and will generate unique resources, biobanks and datasets that will enable mechanistic studies of intestinal biology unobtainable to date.